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Abstract

Abscisic acid (ABA) has been known as a phytohormone of land plants, which is synthesized in response to abiotic stresses and induces various physiological responses, but is also found from eukaryotic algae. Recently, we reported that a unicellular red alga Cyanidioschyzon merolae produced ABA, which prevented cell growth and enhanced salt stress tolerance (Kobayashi et al., 2016). This report describes the detailed method for the extraction and quantification of ABA in the model red alga C. merolae.

The phytohormone ABA has been found in divergent photosynthetic eukaryotes, but the function in unicellular algae remained unclear. In a recent study, we showed that a unicellular red alga C. melorae accumulates ABA in response to salt stress by the present protocol. This is the detail of the first published protocol for the extraction and quantification of ABA from C. merolae. This protocol is optimized for C. merolae based on the land plant protocol.

The optical density (OD) of C. merolae liquid culture is measured by spectrophotometer at 750 nm. When the OD750 reaches 10, cells are diluted to yield an OD750 of approximately 0.5 in 350 ml MA2 medium (Kobayashi et al., 2010). Grow the cells under illumination with fluorescent white light (50 μM photons m-2 s-1) at 42 °C, bubbled with air supplemented with 2% CO2. After incubation for 16 h, measure the OD750, transfer the culture to 500 ml centrifuge bottle, and collect the cells by centrifuging with angle rotor at 3,000 x g for 3 min at room temperature. Gently resuspend the pellet in 350 ml MA2 medium containing 500 mM NaCl and further cultivate for 3 h under the same condition.

The retention time of the single peak areas is recorded and calculate the average of each peak.

Create a calibration curve using the average of each peak. Calculate the ABA amount using a specific standard calibration curve (An example is shown in Figure 2).

Figure 2. ABA standard curve. Standard curve was made based on peak value in HPLC analysis. Example: 300 μl extract was obtained starting from salt stressed cell culture (350 ml, OD750 = 0.8), and 10 μl of the 300 μl was subjected to HPLC analysis, which resulted in the read of 133.533 AU. Based on the calibration curve, the total amount of ABA contained in the starting material was calculated as 68487.5 pmole.

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